家蚕miRNAs对丝胶基因Ser-1、Ser-2的转录后调控研究

MicroRNA(miRNA) as a kind of the significant regulatory factors of gene expression at the post-transcriptional level participates various physiological processes such as development, metabolism, and disease occurrence etc. Researches showed that the miRNAs played regulatory role on fibroin gene. Sericin genes are specifically expressed in the middle silk gland,whether the miRNAs play regulatory role on sericin genes’ expression have not been reported. In this project, the miRNAs which may have potential regulation effects on sericin genes Ser-1 and Ser-2 will be predicted and validated using High-throughput sequencing combined with bioinformatics analysis. Then, the transient expression system at cellular level will be used to illustrate the regulatory role of miRNAs; the recombinant AcMNPV-miRNA report gene system and the methods of restraining endogenous miRNAs applied at individual level will be conducted for further function verification of miRNAs that effect on Ser-1 and Ser-2. Then, the microRNA level for different silkworm strains which contain different content of sericin will be investigated to evaluate the function of microRNA in the regulation of sericin. Therefore, the basis for further elucidation of silk protein synthesis and regulation mechanisms of sericin genes and the interactions with miRNAs will be provided through the research of the regulatory mechanisms between miRNAs and sericin genes. Furthermore, it will provide experimental data and theoretical basis for molecular breeding of silkworm.

MicroRNAs(miRNAs)作为转录后基因表达的一个重要调控因子，参与了包括发育、代谢、疾病发生等各种重要的生理过程，研究显示家蚕miRNAs对丝素蛋白基因有调控作用。丝胶基因是家蚕中部丝腺特异表达的基因，但是miRNAs对丝胶基因表达调控研究尚未见报道，本研究拟通过对家蚕中部丝腺组织RNA高通量测序，结合家蚕miRNA数据库，生物信息学预测参与家蚕丝胶基因Ser-1、Ser-2转录后调控的家蚕miRNAs，利用家蚕培养细胞瞬时表达系统、重组AcMNPV-miRNA报告基因检测系统和反义核酸分子抑制方法在细胞水平和个体水平分析验证候选miRNAs对Ser-1、Ser-2的调控作用，并通过调查不同丝胶含量品种的相关microRNA表达水平来研究microRNA在丝胶表达调控中的功能，为阐明microRNA对家蚕丝蛋白基因表达的调控机制提供实验数据，为优质丝蚕品种的分子育种提供理论基础。

蚕丝蛋白表达调控是一个复杂的网络，家蚕miRNA（bmo-miRNA）参与了蚕丝蛋白基因的转录后调控。本项目利用Solexa测序技术对家蚕P50 与裸蛹（Nd）突变品种5 龄3d 幼虫中部丝腺进行了高通量测序，分析获得了86 个在P50 与Nd 中的表达存在显著差异的miRNAs；生物信息学技术预测获得bmo-miR-2711、bmo-miRNA-275、bmo-miRNA-3377、和bmo-miR-0047等多个对丝胶基因BmSer-1、BmSer-2等具有调控作用的家蚕miRNA。利用RT-PCR技术，分析预测的miRNA和靶基因的表达水平，初步判断它们的调控关系。.为了验证预测miRNA对靶基因的调控作用，以pGL3为载体、家蚕A3启动子驱动的荧光素酶基因（luc）为报告基因，在luc下游连接预测靶基因（BmSer-1和BmSer-2）的3'UTR，构建靶基因表达质粒；以pCDNA3.0为载体，egfp为报告基因，分别构建家蚕miRNA真核表达载体；miRNA表达载体和靶基因3'UTR表达载体共转染BmN细胞，以海肾荧光素酶质粒pRL做内参，检测细胞荧光素酶的活性。细胞水平证实bmo-miR-2711、bmo-miR-0047下调丝胶基因BmSer-1的表达；bmo-miRNA-275能下调BmSer-2的表达。本研究结果有助于更好地理解miRNA的功能，以及miRNA对靶基因的调控作用，为深入研究bmo-miRNA的功能奠定了良好的基础，也为阐明蚕丝蛋白基因表达调控提供了新的实验数据。